86 research outputs found
Molecular mechanisms and physiological significance of organelle interactions and cooperation
No full textThis is the final version of the ebook. Available from Frontiers Media via the DOI in this recor
Seventy years of peroxisome research: current advances and future perspectives
No full textBiotechnology and Biological Sciences Research Council http://dx.doi.org/10.13039/501100000268European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska Curie actionGW4 BioMed MRC Doctoral Training PartnershipDeutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Ruhr-Universität Bochum 50110000625
Nachweis Estrogen-induzierter Genexpression in Fischen und Hepatycytenprimärkulturen als Marker für endokrin wirksame Substanzen in der Umwelt
No full textVitellogenin und Estrogenrezeptoren stehen in der Leber von Fischen unter estrogener Kontrolle und werden in männlichen Individuen nur gering exprimiert. Durch exogene Applikation Estrogen-wirksamer Stoffe wird jedoch auch bei Männchen eine Expression induziert, so dass beide Gene als Marker für estrogene Wirkungen dienen können. Unter dieser Voraussetzung wurde in der vorliegenden Dissertation ein Nachweissystem für die Expression beider Gene in Hepatocytenkulturen aus der Regenbogenforelle entwickelt. In einem ersten Bewertungsansatz wurde das estrogene Potential ausgewählter Xenoestrogene erfasst. Ebenso konnte in einer Stichprobe bei 6 Abwässern aus dem Schweizer Mittelland die Anwendbarkeit der Methode auf komplexe Lösungen nachgewiesen werden. Für eine grundlegende Beurteilung des estrogenen Potentials einer Substanz sind jedoch In vivo-Untersuchungen unerlässlich, da nur die Belastung eines intakten Organismus Aufschluss über Bioakkumulation, metabolische Umsetzung und Exkretion gibt. Zur Etablierung geeigneter Nachweissysteme zur Expression Estrogen-regulierter Gene in den beiden Modellfischarten Medaka und Zebrabärbling wurde zunächst Sequenzinformation über die cDNAs von Vitellogeninen beider Fischarten sowie Estrogenrezeptor des Zebrabärblings ermittelt. Mit Hilfe dieser Daten wurden auf semiquantitativer RT-PCR basierende Nachweissysteme für diese Genprodukte sowie für Estrogenreptor und Choriogenin H des Medakas bzw. ZP2 des Zebrabärblings entwickelt. Anschließend erfolgten vergleichende Studien zur zeitlichen und dosisabhängigen Expression dieser Gene nach Exposition verschiedener Xenoestrogene. Bei der Auswertung der Ergebnisse waren distinkte Unterschiede in der Empfindlichkeit beider Fischarten für die einzelnen Modellsubstanzen festzustellen. Für die Bewertung der Emission endokrin wirksamer Substanzen in das Freiland ergibt sich hieraus die Konsequenz, dass signifikante Artunterschiede in der estrogenen Sensibilität zu berücksichtigen sind
Mover is a homomeric phospho-protein present on synaptic vesicles.
No full textWith remarkably few exceptions, the molecules mediating synaptic vesicle exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates. Mover was found in a yeast-2-hybrid assay using the vertebrate-specific active zone scaffolding protein bassoon as a bait. Peptides of Mover have been reported in proteomics screens for self-interacting proteins, phosphorylated proteins, and synaptic vesicle proteins, respectively. Here, we tested the predictions arising from these screens. Using flotation assays, carbonate stripping of peripheral membrane proteins, mass spectrometry, immunogold labelling of purified synaptic vesicles, and immuno-organelle isolation, we found that Mover is indeed a peripheral synaptic vesicle membrane protein. In addition, by generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a bona fide phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites
Identificação de potenciais alvos para terapias dirigidas ao hospedeiro contra a infeção pelo vírus influenza A
No full textInfluenza A virus (IAV) arises every year as a threat to human health, being the
causative agent of most of the seasonal respiratory epidemics and the major
influenza pandemics in the last century. Influenza, or flu, is associated with high
levels of morbidity and mortality in high-risk populations, such as the elderly
and immunocompromised individuals. Nowadays, IAV spreading and severe
disease can be prevented by annual vaccination and the administration of
antiviral therapeutics that have been designed against viral components to
prevent viral replication. Nevertheless, the continuous emergence of novel
genetic variants or new viral strains of IAV due to its rapid ability to evolve,
hampers the efficacy of virus-directed therapeutics. It is, hence, essential to
study the interactions between IAV and the host cell, in order to further discover
potential cellular factors that can in due course act as targets for the
development of novel host-directed therapies. In this work, we present two
distinct overviews of this interplay that focus on the one hand on a
comprehensive analysis of host cell proteostasis during IAV infection, and, on
the other hand, on the study of the role of peroxisome metabolism on antiviral
defense and IAV propagation.
In the first study, we explored the interplay between IAV and the host’s
proteostasis at different stages within a single infection cycle. Our results
demonstrate that IAV infection induces the accumulation of protein aggregates,
upon high rates of viral protein translation. We also demonstrate the
concomitant deregulation of the IRE1 branch of the unfolded protein response
(UPR), and the attenuation of virus production upon its inhibition. The
accumulation of the virus-induced protein aggregates seems to be further
associated with a virus-induced deregulation of the host cell RNA metabolism.
Moreover, interference with the accumulation of these aggregates by the
presence of a quinoline-steroid compound specifically designed to prevent the
formation of proteins aggregates, strongly hinders the proper formation of new
infectious virus particles. In the second study, we investigated the previously
described anti-viral/pro-viral dual role of peroxisomes during a viral infection.
We have identified specific peroxisomal proteins and mechanisms that are
required for a proper antiviral response against RNA viruses or that play a
critical role during IAV replication. We also showed that the metabolic and
physical interplay with other organelles is essential for the establishment of a
robust innate immune response.
Altogether, both projects allowed us to identify previously undescribed cellular
mechanisms that play important roles not only during IAV replication but also
for the establishment of an efficient antiviral response by the host cell. Further
studies are proposed to follow these results and ultimately uncover novel
targets for innovative host-directed therapeutics with antiviral activity.O vírus da influenza A (IAV) surge todos os anos como uma ameaça para a
saúde humana, sendo o agente causador da maioria das epidemias
respiratórias sazonais e das principais pandemias gripais do último século. A
doença causada por estes vírus está associada a elevados níveis de
morbilidade e mortalidade em populações de alto risco, tais como idosos ou
indivíduos imunocomprometidos. Atualmente, a propagação do IAV e a doença
grave podem ser evitadas através da vacinação anual e da administração de
terapêuticas antivirais concebidas contra componentes virais. No entanto, o
aparecimento contínuo de novas variantes genéticas e novas estirpes virais,
devido à sua rápida capacidade de evolução, dificulta a eficácia de
terapêuticas dirigidas ao vírus. É, assim, essencial estudar as interações entre
o IAV e a célula hospedeira, de forma a descobrir potenciais fatores celulares
que possam atuar como alvos para o desenvolvimento de novas terapias
dirigidas ao hospedeiro. Neste trabalho, apresentamos duas visões distintas
desta interação que se centram, por um lado, numa análise abrangente da
proteostase da célula hospedeira durante a infeção por IAV e, por outro lado,
no estudo do papel do metabolismo dos peroxissomas na defesa antiviral e na
propagação do IAV.
No primeiro estudo, explorámos a interação entre o IAV e a proteostase do
hospedeiro em diferentes fases do ciclo de infeção. Os nossos resultados
demonstram que a infeção induz a acumulação de agregados proteicos, assim
como a desregulação do ramo IRE1 da resposta a proteínas mal enoveladas
(UPR) e a atenuação da produção do vírus após a respetiva inibição. A
acumulação dos agregados proteicos parece estar associada a uma
desregulação induzida pelo vírus do metabolismo do ARN da célula
hospedeira. A interferência com a acumulação destes agregados pela
presença de um composto hibrido quinolina-esteroide, especificamente
concebido para impedir a formação de agregados de proteínas, impede
fortemente a formação adequada de novas partículas virais infeciosas. No
segundo estudo, investigámos o papel antiviral e pro-viral dos peroxissomas
durante as infeções virais. Identificámos proteínas e mecanismos
peroxissomais específicos que são necessários para uma resposta antiviral
adequada contra vírus de ARN, ou que desempenham um papel crítico
durante a replicação de IAV. Mostrámos também que a interação metabólica e
física com outros organelos é essencial para o estabelecimento de uma
resposta imune inata robusta.
Em conjunto, ambos os projetos permitem identificar mecanismos celulares
anteriormente não descritos que desempenham papéis importantes não só
durante a replicação do IAV, mas também para o estabelecimento de uma
resposta antiviral eficiente por parte da célula hospedeira. São propostos
estudos futuros que permitam levar à descoberta novos alvos para
terapêuticas antivirais inovadoras dirigidas ao hospedeiro.Programa Doutoral em Biomedicin
Isolation of Peroxisomes from Rat Liver and Cultured Hepatoma Cells by Density Gradient Centrifugation
No full text
Detection and immunolabelling of peroxisomal proteins
No full textThis is the author accepted manuscript. The final version is available from Humana Press via the DOI in this record.Peroxisomes are essential organelles in mammals which contribute to cellular lipid metabolism and redox homeostasis. The spectrum of their functions in human health and disease is far from being complete, and unexpected and novel roles of peroxisomes are being discovered. To date, those include novel biological roles in anti-viral defence, as intracellular
signalling platforms and as protective organelles in sensory cells. Furthermore, peroxisomes are part of a complex network of interacting subcellular compartments which involves metabolic cooperation, cross-talk and membrane contacts. As potentially novel peroxisomal proteins are continuously discovered, there is great interest in the verification of their peroxisomal localisation. Here, we present protocols used successfully in our laboratory for the detection and immunolabelling of peroxisomal proteins in cultured mammalian cells. We present immunofluorescence and fluorescence-based techniques as well as reagents to determine peroxisome-specific targeting and localisation of candidate proteins.We would like to thank A. Manner for providing images for Fig. 1D. This work was supported by the Marie Curie Initial Training Network (ITN) action (FP7-2012-PERFUME-316723) and the Biotechnology and Biological Sciences Research Council (BB/K006231/1; BB/N01541X/1)
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